DE4414940C2 - Luminescence scanning microscope with two photons excitation - Google Patents
Luminescence scanning microscope with two photons excitationInfo
- Publication number
- DE4414940C2 DE4414940C2 DE4414940A DE4414940A DE4414940C2 DE 4414940 C2 DE4414940 C2 DE 4414940C2 DE 4414940 A DE4414940 A DE 4414940A DE 4414940 A DE4414940 A DE 4414940A DE 4414940 C2 DE4414940 C2 DE 4414940C2
- Authority
- DE
- Germany
- Prior art keywords
- array
- scanning
- luminescence
- detector
- semiconductor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Revoked
Links
- 238000004020 luminiscence type Methods 0.000 title claims description 18
- 230000005284 excitation Effects 0.000 title claims description 10
- 239000004065 semiconductor Substances 0.000 claims description 14
- 230000035945 sensitivity Effects 0.000 claims description 3
- 238000003491 array Methods 0.000 claims 1
- 238000011144 upstream manufacturing Methods 0.000 claims 1
- 238000005259 measurement Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
Description
Die Erfindung betrifft ein Lumineszenz-Rastermikroskop gemäß dem Oberbegriff des Hauptanspruches.The invention relates to a scanning luminescence microscope according to the preamble of the main claim.
Ein derartiges Rastermikroskop mit einer Laser-, einer Filter- und einer Detektoranordnung ist aus der US 5034613 bekannt. Die Laseranordnung regt die Lumineszenz einer zu untersuchen den Probe an. Die Filteranordnung separiert das Lumineszenz licht der Probe vom Laserlicht und die Detektoranordnung registriert das Lumineszenzlicht.Such a scanning microscope with a laser, a filter and a detector arrangement is known from US 5034613. The laser arrangement stimulates the luminescence to be examined the rehearsal. The filter arrangement separates the luminescence light of the sample from the laser light and the detector arrangement registers the luminescent light.
Die DE 40 35 799 A1 beschreibt ein Rastermikroskop, bei dem meh rere Rasterpunkte gleichzeitig angeregt und mit einem Detek torarray konfokal registriert werden. Dieses Mikroskop arbeitet ohne zwei Photonen Anregung.DE 40 35 799 A1 describes a scanning microscope in which meh other halftone dots simultaneously excited and with a detec torarray can be registered confocally. This microscope works without two photons excitation.
Aus der DE 35 05 728 C2 ist ferner ein Verfahren zur gezielten Zerstörung unerwünschter Nukleinsäuren in der unmittelbaren Nachbarschaft von Proteinen bekannt, bei dem die Probe einer zwei Photonen Anregung unterworfen wird. Dabei wird das zu un tersuchende Material zerstört. DE 35 05 728 C2 also describes a method for targeted Destruction of unwanted nucleic acids in the immediate Neighborhood of proteins known in which the sample is a is subjected to two photons excitation. This becomes too un investigative material destroyed.
Die Druckschrift "Proceedings Spie: Three Dimensional Micros copy: Image Acquisition and Processing, vol. 2184, 7 February 1994, San Jose USA; pages 66-71, Hänninen and Hell" beschreibt eine Vorgehensweise bei der die Spitzenleistung erniedrigt und zugleich die Pulsdauer vergrößert wird, so daß sie < 1 ps ist. Dadurch sollen komplexe und teure fs Laser umgangen werden.The publication "Proceedings Spie: Three Dimensional Micros copy: Image Acquisition and Processing, vol. 2184, February 7 1994, San Jose USA; pages 66-71, Hänninen and Hell " a procedure in which the peak performance lowers and at the same time the pulse duration is increased so that it is <1 ps. This is intended to circumvent complex and expensive fs lasers.
Aufgabe der vorliegenden Erfindung ist es, ein Lumineszenz- Rastermikroskop mit zwei Photonen Anregung anzugeben, das ko stengünstig herstellbar ist und schnell und probenschonend ar beitet.The object of the present invention is to provide a luminescence Scanning microscope with two photons excitation to indicate the ko is inexpensive to manufacture and quick and gentle on samples works.
Diese Aufgabe wird durch ein Lumineszenz-Rastermikroskop mit den Merkmalen des Anspruchs 1 gelöst. Dabei erzeugt eine Laseranordnung als Array von Halbleitern Lichtimpulse größer als 1 Picosekunde und die Detektoranordnung ist als Array von Halbleiterdetektoren ausgebildet. Durch die Anregung mit Laserimpulsen, deren Dauer größer ist als eine Pikosekunde oder durch die Anregung mit kontinuierlichem Licht sind die Leistungsspitzen im Anregungslicht soweit reduziert, daß eine Zerstörung des Objektes nicht auftritt. Da die verwendeten Laserimpulse eine niedrigere Leistung aufweisen, als die Leistung im Sub-Picosekundenbereich, sind längere Meßzeiten in der Regel angebracht, wobei die Empfindlichkeit des verwendeten Detektors den Meßbedingungen angepaßt wird. Es hat sich herausgestellt, daß wenn als Auswertungsverfahren das Verfahren des Photonenzählens eingesetzt wird, dieses zu sehr guten Messergebnissen führt. Besonders vorteilhaft ist es, wenn Halbleiterlaserarray und das Detek torhalbleiterarray bezüglich des Objektivs des Rastermikroskops in zueinander konjugierten Positionen angeordnet sind. Sogar mit kontinuierlichem Laserlicht mittlerer Leistung ist die Aufnahme und Auswertung der Meßergebnisse möglich.This task is performed using a scanning luminescence microscope solved the features of claim 1. A laser arrangement generates an array of semiconductors Light pulses greater than 1 picosecond and the detector array is designed as an array of semiconductor detectors. Through the Excitation with laser pulses, the duration of which is longer than one Picosecond or by excitation with continuous light the power peaks in the excitation light are reduced so far that the object is not destroyed. Since the used laser pulses have a lower power, than the performance in the sub-picosecond range are longer Measurement times are usually appropriate, with sensitivity of the detector used is adapted to the measurement conditions. It it has been found that if the evaluation method is Methods of photon counting used this leads to very good measurement results. Especially It is advantageous if the semiconductor laser array and the detector Gate semiconductor array with respect to the lens of the scanning microscope are arranged in conjugate positions. Even with continuous laser light of medium power is the Recording and evaluation of the measurement results possible.
Es ist vorteilhaft, wenn ein Detektorhalbleiterarray mit einem hohen Signal-Rausch-Verhältnis eingesetzt wird. Von besonderer Bedeutung ist dabei, daß der eingesetzte Detektor eine entsprechend hohe Empfindlichkeit und ein kleines Eigenrauschen aufweist. Durch die arrayförmige Anordnung der Laser und Detektoren und die dadurch gegebene Bestrahlung und Messung von mehreren Rasterpunkten, ist die Möglichkeit gege ben, die Dauer des Verfahrens zu verkürzen.It is advantageous if a detector semiconductor array with a high signal-to-noise ratio is used. Of special It is important that the detector used is a accordingly high sensitivity and a small one Has inherent noise. Due to the array-like arrangement of the Lasers and detectors and the resulting radiation and Measurement of several grid points is possible ben to shorten the duration of the procedure.
Bei dem erfindungsgemäßen Luminesenz-Rastermikroskop sind weiterhin Filter vorgesehen zum Separieren des von der Probe emitierten Lichtes von dem Laserlicht. Anstatt eines Filters kann auch ein Detektor eingesetzt werden, der die entsprechende Wellenlänge herausfiltert.In the luminescence scanning microscope according to the invention furthermore filters are provided for separating the sample emitted light from the laser light. Instead of a filter a detector can also be used, which detects the filters out the corresponding wavelength.
Das Detektorhalbleiterarray kann auch als Photonenzähler aus gebildet sein.The detector semiconductor array can also function as a photon counter be educated.
Eine schematische Darstellung eines Lumineszenz-Rastermikro skopes zur Durchführung des erfindungsgemäßen Verfahrens ist in der einzigen Figur wiedergegeben.A schematic representation of a luminescence scanning micro scopes for performing the method according to the invention reproduced in the single figure.
Das Lumineszenz-Rastermikroskop weist einen Laser 1 zur Erzeugung des Laserlichtes, einen Fotodedektor 2 zur Auswertung der Meßergebnisse und ein Objektiv 3 zur Fokussierung des von der Laserlichtquelle 1 ausgesandten Laserstrahles 4 auf das zu untersuchende Objekt 5 auf. Der Laserstrahl 4 wird mit einer Strahlrastereinrichtung 6 zum Abrastern des Objektes 5 gesteuert. Ferner weist das Lumineszenz-Rastermikroskop Filter 7 und 8 zum Separieren des Laserlichtes 4 vom Lumineszlicht auf. Gemäß der Erfindung ist die Laserlichtquelle 1 als ein herkömmlicher Laser mit kontinuierlicher oder gepulster Strahlung ausgebildet. Die Laserlichtquelle 1 kann auch durch eine arrayförmige Anordnung von Lasern gebildet sein. Das gleiche trifft für den Fotodedektor 2 zu. Dieser kann entweder punkt- oder arrayförmig ausgebildet sein. Im Falle einer arrayförmigen Anordnung der Laser 1 und Detektoren 2 sind diese sowie der abzubildende Probenbereich in jeweils zueinan der optisch konjugierten Ebenen zur gleichzeitigen Aufnahme von mehreren Rasterpunkten angeordnet.The scanning luminescence microscope has a laser 1 for generating the laser light, a photo detector 2 for evaluating the measurement results and a lens 3 for focusing the laser beam 4 emitted by the laser light source 1 onto the object 5 to be examined. The laser beam 4 is controlled with a beam raster device 6 for scanning the object 5 . Furthermore, the luminescence scanning microscope has filters 7 and 8 for separating the laser light 4 from the luminescent light. According to the invention, the laser light source 1 is designed as a conventional laser with continuous or pulsed radiation. The laser light source 1 can also be formed by an array-like arrangement of lasers. The same applies to the photo detector 2 . This can be either point-shaped or array-shaped. In the case of an array-shaped arrangement of the lasers 1 and detectors 2 , these and the sample area to be imaged are arranged in each case in the optically conjugate planes for the simultaneous recording of several raster points.
Claims (6)
- - mit einer Laseranordnung (1) zur Zwei-Photonen-Anregung von dreidimensional in einem zu beobachtenden Objekt (5) verteilten, lumineszierenden Molekülen,
- - mit einer das zu beobachtende Objekt (5) abtastenden Rastereinrichtung (6),
- - und mit einem Strahlteiler (7) zwischen dem Objekt (5) und
einer Detektoranordnung (2), der das Lumineszenzlicht der
Lumineszenzmoleküle zu der Detektoranordnung (2) auskoppelt,
dadurch gekennzeichnet, - - daß die Laseranordnung (1) die Zwei-Photonen-Anregung mit Lichtpulsen von einer Dauer größer als 1 Pikosekunde erzeugt,
- - und daß die Laseranordnung (1) als Array von Halbleiterlasern und die Detektoranordnung (2) als Array von Halbleiterdetektoren ausgebildet ist,
- - wobei diese beiden Arrays das Objekt abtasten.
- with a laser arrangement ( 1 ) for two-photon excitation of luminescent molecules distributed three-dimensionally in an object ( 5 ) to be observed,
- - With a raster device ( 6 ) scanning the object to be observed ( 5 ),
- and with a beam splitter ( 7 ) between the object ( 5 ) and a detector arrangement ( 2 ), which couples the luminescent light of the luminescent molecules to the detector arrangement ( 2 ),
characterized by - that the laser arrangement ( 1 ) generates the two-photon excitation with light pulses of a duration greater than 1 picosecond,
- - and in that the laser array (1) is formed as an array of semiconductor lasers and the detector arrangement (2) as an array of semiconductor detectors,
- - whereby these two arrays scan the object.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE4414940A DE4414940C2 (en) | 1994-04-28 | 1994-04-28 | Luminescence scanning microscope with two photons excitation |
EP95916571A EP0706671A1 (en) | 1994-04-28 | 1995-04-27 | Luminescence scanning microscopy process and a luminescence scanning microscope |
US08/571,839 US5777732A (en) | 1994-04-28 | 1995-04-27 | Luminescence-scanning microscopy process and a luminescence scanning microscope utilizing picosecond or greater pulse lasers |
PCT/DE1995/000566 WO1995030166A1 (en) | 1994-04-28 | 1995-04-27 | Luminescence scanning microscopy process and a luminescence scanning microscope |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE4414940A DE4414940C2 (en) | 1994-04-28 | 1994-04-28 | Luminescence scanning microscope with two photons excitation |
Publications (2)
Publication Number | Publication Date |
---|---|
DE4414940A1 DE4414940A1 (en) | 1995-11-02 |
DE4414940C2 true DE4414940C2 (en) | 1998-07-02 |
Family
ID=6516737
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
DE4414940A Revoked DE4414940C2 (en) | 1994-04-28 | 1994-04-28 | Luminescence scanning microscope with two photons excitation |
Country Status (4)
Country | Link |
---|---|
US (1) | US5777732A (en) |
EP (1) | EP0706671A1 (en) |
DE (1) | DE4414940C2 (en) |
WO (1) | WO1995030166A1 (en) |
Cited By (3)
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DE10314750A1 (en) * | 2003-03-31 | 2004-11-04 | Leica Microsystems Heidelberg Gmbh | Scanning microscope for biological applications has an objective with a contrast device which enables use of the microscope in a Hoffman-modulation contrast mode |
US9846121B2 (en) | 2009-06-17 | 2017-12-19 | W.O.M. World Of Medicine Gmbh | Device and method for multi-photon fluorescence microscopy for obtaining information from biological tissue |
CN109718476A (en) * | 2018-12-28 | 2019-05-07 | 中国科学院苏州生物医学工程技术研究所 | 3-D scanning two-photon stimulating system and its stimulating method |
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DE19653413C2 (en) | 1996-12-22 | 2002-02-07 | Stefan Hell | Scanning microscope, in which a sample is simultaneously optically excited in several sample points |
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US5034613A (en) * | 1989-11-14 | 1991-07-23 | Cornell Research Foundation, Inc. | Two-photon laser microscopy |
DE4035799A1 (en) * | 1990-11-10 | 1992-05-14 | Zeiss Carl Fa | Confocal scanning microscope with computer control - has illumination raster corresponding to raster of CCD sensor receiving image of scanned object |
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-
1994
- 1994-04-28 DE DE4414940A patent/DE4414940C2/en not_active Revoked
-
1995
- 1995-04-27 US US08/571,839 patent/US5777732A/en not_active Expired - Lifetime
- 1995-04-27 WO PCT/DE1995/000566 patent/WO1995030166A1/en not_active Application Discontinuation
- 1995-04-27 EP EP95916571A patent/EP0706671A1/en not_active Ceased
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DE3505728C2 (en) * | 1984-02-16 | 1989-01-19 | Molecular Biophysics Technology, Inc., Philadelphia, Pa., Us | |
US5034613A (en) * | 1989-11-14 | 1991-07-23 | Cornell Research Foundation, Inc. | Two-photon laser microscopy |
DE4035799A1 (en) * | 1990-11-10 | 1992-05-14 | Zeiss Carl Fa | Confocal scanning microscope with computer control - has illumination raster corresponding to raster of CCD sensor receiving image of scanned object |
Non-Patent Citations (1)
Title |
---|
Proceedings SPIE: Three Dimensional Microscopy, Image Acquisition and Processing, Two Photon Excitation in Time-Resolved FluorescensMikroscopy/vol. 2184, 7. Feb. 1994, San Jose USA, pp 66-71/ Pekka Hänninen and Stefan Hell * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10314750A1 (en) * | 2003-03-31 | 2004-11-04 | Leica Microsystems Heidelberg Gmbh | Scanning microscope for biological applications has an objective with a contrast device which enables use of the microscope in a Hoffman-modulation contrast mode |
US9846121B2 (en) | 2009-06-17 | 2017-12-19 | W.O.M. World Of Medicine Gmbh | Device and method for multi-photon fluorescence microscopy for obtaining information from biological tissue |
CN109718476A (en) * | 2018-12-28 | 2019-05-07 | 中国科学院苏州生物医学工程技术研究所 | 3-D scanning two-photon stimulating system and its stimulating method |
Also Published As
Publication number | Publication date |
---|---|
EP0706671A1 (en) | 1996-04-17 |
WO1995030166A1 (en) | 1995-11-09 |
DE4414940A1 (en) | 1995-11-02 |
US5777732A (en) | 1998-07-07 |
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